In the cultivation of mammal cells in vitro, the basal medium is traditionally supplemented with mammal blood serum, which contains several partly unknown cell growth promoting agents, such as growth factors, vitamins, trace elements, hormones, binding proteins and attachment factors. A serum suitable for most purposes is fetal bovine serum, the price of which has increased greatly with increasing demand in recent years. Another reason for the high price is the limited availability of sufficiently pure serum. Besides the high price, another problem with serum is its complex composition and especially the high protein concentration, which hampers the isolation of produced substances from the culture medium. There is also a high risk of contamination when using serum as a supplement in media. Both technologically and economically, monoclonal antibody production forms an important part of animal cell culture technology. Monoclonal antibodies are produced in large quantities for a variety of clinical and scientific purposes. In large-scale production of monoclonal antibodies, it is important to use a suitable culture medium. Antibody production should be continuous and reproducible; process costs should be as low as possible; antibodies should be readily purifiable; and contamination caused by microbes and endotoxins should be avoided. The same applies to the production of other biological substances, such as growth factors, hormones and vaccines, which are produced for clinical purposes by culturing genetically engineered animal cells. It is, however, very difficult to meet these requirements when using fetal bovine serum as a cell culture supplement.
Therefore, attempts have been made to find alternatives for the use of serum by developing various basal media and serum substitutes. They contain completely or partly purified growth promoting agents produced biosynthetically or isolated directly from a biological substance. Such growth promoting agents, e.g. various peptide growth factors such as insulin and insulin-like growth factors (IGF-1 and IGF-2), are not only present in serum but also in bovine colostrum. The synthesis, isolation and purification of growth factors are usually difficult to carry out, and literature does not teach any methods suitable for large-scale production. The use of purified growth factors is further limited by their high cost, which is even higher than that of fetal serum.
It is known from literature to use whey fractions obtained as a by-product in the production of milk and cheese as a supplement in cell culture media. However, the growth promoting activity of milk decreases sharply after calving, being very low, almost negligible, as soon as three days after calving as well as at subsequent lactation stages. On the other hand, milk possibly contains other cell growth promoting agents, nutrients, etc, which may promote the growth of cells.
It is also known that bovine colostrum and its fractions promote the growth of mammal cells in vitro. In addition to numerous components essential for cell growth, bovine colostrum, however, contains high amounts of immunoglobulins, mainly IgG, and other proteins, such as casein micelles, .alpha.-lactalbumin, .beta.-lactoglobulin, and albumin. Immediately after lactation the IgG concentration may be as high as 40 to 60 g/l. This is a clear disadvantage when colostrum is used as such for the cultivation of hybridoma cultures, since it makes the isolation and purification of hybridoma products more difficult.
Another serious problem is associated with psychrotrophic microorganisms, mainly gram-negative bacteria, which are the most common spoilers of milk during storage. Lipopolysaccharides (endotoxins) produced by microorganisms are responsible for many pathophysiological effects accompanying infections caused by gram-negative-bacteria. Thus endotoxins are extremely harmful contaminants, and their removal is essential especially when the substances produced in cell culture are intended for human use.
EP Patent Application 219 372, Linden et al., describes fractions of ordinary milk having a certain molecular weight, their preparation and use in cell culture media. This patent document teaches that milk is first ultracentrifuged, which is technically difficult to carry out in large-scale purification. Fractionation itself is then carried out by ultrafiltration based on the different molecular sizes of the substances and having a low resolution capacity. Accordingly, the protein concentration of the final product is very high in relation to the growth promoting activity. The inventors also describe the use of whey or whey fractions obtained as a by-product in the production of cheese in the cultivation of mammal cells (Biotechnology Techniques 2 (1988) p. 253-258, Damerdhjii et al., and Lait 70 (1990) p. 313-324, Derouiche et al.). The problem here is also the very high protein concentration and the risk of chemical and microbiological contamination.
EP Patent Application 313 515, Burk, R. R. & Cox, D., describes a polypeptide growth factor in milk, processes for separating and purifying it from milk and milk products, and its use as a pharmaceutical, dietary additive, and cell culture media supplement. The process of this patent document comprises cation exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, and polyacrylamide gel electrophoresis. As already mentioned above, the growth promoting activity of milk decreases sharply after calving. However, colostrum and its treatment are not referred to in this patent document.
U.S. Pat. No. 4,440,860, Klagsbrun, describes cell culture media containing milk or colostrum and fibronectin, and the preparation of such media. It is recited that the fractions are prepared by gel filtration and isoelectric focusing, which is not suitable for large-scale production. It is not disclosed in the patent document how the microbiological purity of the final product is ensured. Instead of comparing the growth promoting activity of the fractions described in the patent document with fetal bovine serum, the most common type of serum used in cell culture media, it is compared with calf serum having a substantially lower activity. The endotoxin concentration of the fractions is not mentioned at all. Furthermore, it is to be noted that it is assumed in the patent document that the growth factor fractions of the colostrum of different mammal species are similar. However, further research has shown that the principal growth factors of e.g. bovine and human colostrum deviate from each other to such an extent that they cannot be isolated by the same method (Endocrinology 115 (1984) p. 273-282, Shing, Y. W. & Klagsbrun, M.).
In Methods in Enzymology, vol. 146, edited by Barnes D. and Sirbasku, D. A., Shing et al. describe a process of purifying a bovine colostrum growth factor (BCGF) based on cation exchange, isoelectric focusing and high-resolution exclusion chromatography. The method is not suitable for growth factor isolation on industrial scale.
Francis et al. have isolated and characterized insulin-like growth factors IGF-1 and IGF-2 from bovine colostrum by a process described in Biochem. J. 251 (1988) p. 95-103. These growth factors have a molecular weight of 8,000 and 7,000 D, respectively, and they are nearly identical in structure with the corresponding human growth factors. This complicated process comprises many steps, such as several cation exchange chromatography and reversed-phase HPLC steps, and so it is not suitable for large-scale production.
The known methods thus all comprise many steps and are difficult to carry out and unsuitable for large-scale production. Complicated and time-consuming purification operations also increase the product cost. It is also to be noted that it is not disclosed in the cited documents how the microbiological purity of the final product is ensured. Neither do they mention the endotoxin concentration of the final product. Endotoxins and problems caused by them are not described in the cited documents, so they do not either suggest any solution to these problems.
The major drawbacks of colostrum when used as such as culture media supplement are its high protein and IgG concentration and usually the high endotoxin concentration. Viruses present in colostrum may also inhibit cell growth or even kill cells. The high protein concentration of colostrum and the insoluble sediments contained in it also hamper the treatment of colostrum and make it difficult if not impossible to sterilize e.g. defatted colostrum through microfilters. If high amounts of defatted colostrum are added to a culture medium, precipitations will occur in the medium.